ISSN 0003-6838, Applied Biochemistry and Microbiology, 2009, Vol. 45, No. 6, pp. 642647. Pleiades Publishing, Inc.

., 2009. Original Russian Text T.A. Misharina, M.B. Terenina, N.I. Krikunova, 2009, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2009, Vol. 45, No. 6, pp. 710716.

Antioxidant Properties of Essential Oils

T. A. Misharina, M. B. Terenina, and N. I. Krikunova
Emanuel Institute of Biochemical Physics, Russian Academy of Sciences ul. Kosygina 4, Moscow, 119991 Russia e-mail: Tmish@rambler.ru
Received September 23, 2008

AbstractBy the method of capillary gas-liquid chromatography, we studied the antioxidant properties and stability during the storage of hexane solutions of 14 individual essential oils from black and white pepper (Piper nigrum L.), cardamom (Elettaria cardamomum L.), nutmeg (Myristica fragrans Houtt.), mace (Myristica fragrans Houtt), juniper berry (Juniperus communis L.), fennel seed (Foeniculum vulgare Mill., var. dulce Thelling), caraway (Carvum carvi L.), dry cinnamon leaves (Cinnamomum zeylanicum Bl.), marjoram (Origanum majorana L.), laurel (Laurus nobilis L.), ginger (Zingiber ofcinale L.), garlic (Allium sativum L.), and clove bud (Caryophyllus aromaticus L.). We assessed the antioxidant properties by the oxidation of aliphatic aldehyde (trans-2-hexenal) into the corresponding carbonic acid. We established that essential oils of garlic, clove bud, ginger and leaves of cinnamon have the maximal efciency of inhibiting hexenal oxidation (80!93%), while black pepper oil has the minimal (49%). Antioxidant properties of essential oils with a high content of substituted phenols depended poorly on their concentrations in model systems. We studied the changes in the composition of essential oils during the storage of their hexane solutions for 40 days in light and compared it with the stability of essential oils stored for a year in darkness. DOI: 10.1134/S000368380906012X

To protect from infections and parasites and in response to stress, plants synthesize low molecular volatile terpenoids, the mixture of which is called essential oils. Essential oils of many plants possess a strong and pleasant aroma and are also biologically active [13]. Due to these properties, essential oils are actively used in the medical and pharmaceutical industries, in aromatherapy and cosmetology, and in the production of natural food avorings[3, 4]. As a rule, essential oils are a complex mixture of organic substances with different functional groups, mainly terpenoids. The composition of essential oils determines their organoleptic properties and biological activity including antioxidant [58]. The study of individual components of different essential oils has shown that many terpenes possess antiradical and antioxidant activity (AOA) and the activity of cyclic monoterpene hydrocarbons with two double bonds is comparable with AOA of phenol and tocopherol [911]. Thus, - and -terpinenes and sesquiterpene hydrocarbons, including caryophyllene, inhibited methyl linoleate oxidation [1013]. Sabinene, - and -terpinenes and - terpinolene are active donors of hydrogen in reference to the 2,2-diphenyl-1-picrilhydrazyl radical, which is used for the estimation of antiradical activity [10, 1416]. Citronellal, neral, and geranial, which are included in many essential oils, possess antiradical activity [10, 14, 17, 18]. As a rule, antioxidant activity of essential oils is higher than for their individual components. This indicates the existence of synergetic effects due to the complex multicomponent composition of oils [15, 19, 20].

To estimate the antioxidant properties of substances or their mixtures, many different methods are used. It was showed that the value of AOA signicantly depends on the method of its estimation, which is why the literature data obtained by different methods are practically incomparable. In addition, the antioxidant properties of essential oils depend on the qualitative and quantitative composition of systems under test [6, 10, 13, 20, 21]. One of the simple and informative methods of quantitative assessment of AOA is based on inhibition of the rate of lower aldehyde autooxidation, for example, hexanal, in the presence of antioxidant substances [7, 8, 20, 22]. This method has been used successfully for to estimate and compare the antioxidant properties of a number of essential oils [20, 22 25]. The aim here is to study and compare antioxidant properties of 14 essential oils in the model system of autooxidation of 2-hexenal, assess the inuence of the composition of essential oils on their AOA, and to study the changes in the composition of essential oils in the process of autooxidation in solutions and in pure forms. METHODS We studied fresh samples of essential oils of black and white pepper (Piper nigrum L.), cardamom (Elettaria cardamomum L.), nutmeg (Myristica fragrans Houtt.), mace (Myristica fragrans Houtt.), juniper berry (Juniperus communis L.), fennel seed (Foeniculum vulgare Mill., var. dulce Thelling), caraway (Carvum carvi L.),

642

ANTIOXIDANT PROPERTIES OF ESSENTIAL OILS

643

dry leaves cinnamon (Cinnamomum zeylanicum Bl.), marjoram (Origanum majorana L.), laurel (Laurus nobilis L.), ginger (Zingiber ofcinale L.), garlic (Allium sativum L.), and clove bud (Caryophyllus aromaticus L.) (company Plant Lipids Ltd., India). To estimate the antioxidant properties of essential oils and their mixture, 200 ml of n-hexane were soluted by 600 l trans-2-hexenal (3 l/ml) and 400 l undecane (2 l/ml), which was an internal standard. The solution was separated into 3-ml aliquots, which were placed in 5-ml glass vials and then 10 l (3.33 l/ml), 50 l (16.5 l/ml) or 210 l (70 l/ml) of essential oils were added. Oil was not added in the control sample. Each sample was prepared twice; the control sample was prepared three times. Samples were stored in vials with stoppers in light under room temperature for 40 days. Every week vials were opened and blown with 10 ml of air with a pipette. The quantitative content of 2-hexenal and components of essential oils in vials were determined by capillary gas chromatography every 10 days from the beginning of storage. Gas-chromatography analysis of the essential oils samples and control sample was conducted on a Kristall 2000M chromatograph (Russia) with a ame ionization detector and an SPB-1 silica capillary column (50 m 0.32 mm, phase layer 0.25 m). The analysis of samples was conducted with the programming of the column temperature from 60 to 250C at a rate of 8C/min under the temperature of detector and injector at 250C. The rate of the carrier gas, helium, through the column was 1.5 ml/min. We analyzed 2 l of hexane solutions at once. The identication of components in oil samples was carried out on the basis of retention indices by their comparison with the literature [26] or experimental data obtained by us. The quantitative content of 2-hexenal and components of essential oils was calculated by the ratio of peak areas, which corresponded to the substances and internal standard. The extent of oxidation of 2-hexenal and essential oils components (%) was determined in reference to their content in initial samples. RESULTS AND DISCUSSION The oils that we chose contained many common components but differed in their quantitative and qualitative composition. We used capillary gas chromatography, which is why we could estimate changes in the content of each oil component in two to three model systems with different oil concentrations and in pure oil, as well as distinguish the oxidation products of the main components. Furthermore, due to the comparison of oil composition and the oxidation rates of components, we managed to reveal some regularity, which enables us to predict and regulate oil composition to obtain stable mixtures. This is very important because essential oils are currently widely used in industry and medicine. Usually the recommended storage time for oils is 1 year; however, nobody has studied yet what
APPLIED BIOCHEMISTRY AND MICROBIOLOGY

AOA, % 100 80 60 40 20 0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Control C1 C2 C3

Fig. 1. Relative antioxidant activity of essential oils; (1) control (2-hexenale), (2) garlic, (3) clove bud, (4) cinnamon leaves, (5) ginger, (6) nutmeg, (7) mace, (8) laurel, (9) marjoram, (10) caraway, (11) fennel, (12) juniper berry, (13) black pepper, (14) white pepper, and (15) cardamom. Concentration of essential oils (l/ml): C1,3.3; C2, 16.5; and C3, 70.

really happens to oils during this period. Earlier we studied the changes in the composition of coriander, laurel, marjoram, and fennel oils during storage of pure oil amples away from light and in light but in bottles of dark glass [2729]. It was established that the main process is oxidation of the oil components. Thus, we found, and then it was proved in [10, 11], that cyclic monoterpene hydrocarbons - and -terpinenes are completely oxidized into aromatic hydrocarbon pcymene. We also found oxidation products of other components of essential oilsoxides, alcohols, and aldehydes [2629]. To estimate the antioxidant properties of essential oils, we used a model system of 2-hexenal daylightinduced autooxidation, which has been widely used in similar studies [7, 8, 2025]. As a criterion for the estimation of antioxidant properties of essential oils, we used a quantity of 2-hexenal, which remained unoxidized after 40 days in regards to the initial quantity (%). Figure 1 presents the results of relative AOA values of the 14 essential oils studied. The model system included garlic, cinnamon leaves, marjoram, clove bud, nutmeg, laurel, or white pepper in two concentrations: 3.3 l/ml and 16.5 l/ml. The quantity of 2-hexenal was 3 l/ml. For the essential oils of ginger, juniper berry, fennel, black pepper, caraway, and cardamom, we studied an additional third system in which the content of oil was 70 l/ml of hexane solution. As is clearly seen from Fig. 1, there was a dependence between concentration and activity for all oils with the exception of garlic and clove bud oils. It is noteworthy that the increase in activity was usually not in proportion to the growth of essential oil concentration. The systems with miniNo. 6 2009

Vol. 45

644 % 120

MISHARINA et al.

110

100 3

90

changes in content of the main polysuldes during the storage of the hexane solution of the essential oil of garlic for 2 months. As can be seen, during this time the quantity of polysuldes increased, while that of threeand tetrasuldes decreased; that is, it happened disproportionally. In the case of the longer storage period of pure oil, such changes were noticed after 6 months away from light and 4 months in light. Also, except for disproportionality, we revealed polysulde oxidation with the formation of sulfoxides and sulfonic acids. As a result, such oil was of no use. The inhibition of 2-hexenal oxidation by clove bud oil was also hardly depended on the concentration and was 7578%. The essential oil of cinnamon leaves preserved 2-hexenal at 6678% (Fig. 1), while the concentration increased by ve times. This oil differed from clove bud oil by its more complex composition. It contained approximately 10% of monoterpene hydrocarbons and alcohols, cinnamaldehyde, and cinnamyl acetate. The content of the main component eugenol was less by 5% and the content of eugenyl acetate was more by 7% than in clove bud oil. AOA and composition stability of both oils were high. In hexane solution, both oils did not change their composition for 60 days. Pure individual oils may also be stable when stored away from light for 2 years and in light for 8 months. In both oils, we did not notice oxidation even of terpene hydrocarbons. The AOA of the essential oils of nutmeg and mace increased from 53 to 69% and from 63 to 78% accordingly, with an increase in their concentration in model solutions (Fig. 1). These oils were close in the quantitative and qualitative content of components. The main compounds in oils were monoterpene hydrocarbons and alcohol 4-terpineol. The aroma of this spice is due to phenol derivativessafrol, myristicin, and elemicinthe content of which in oil was from 0.9 to 2.6%. These compounds together with terpene hydrocarbons corresponded to the AOA of oils. During the autooxidation of oils in hexane solutions for 40 days, only the content of - and -terpinenes decreased by three to four times and the content of their oxidation product, pcymene, increased. In the process of storage of these oils in pure form for 4 months, their content did not change, but storage for 1 year led to the almost complete oxidation of - and -terpinenes and the oxidation of 50% of caryophyllene with the formation of caryophyllene oxide. The AOA of the essential oil of ginger was studied for three model systems with oil concentrations of 3.3, 16.5, and 70 l/ml. In the rst system, oil inhibited the oxidation of 2-hexenal by 54%; in the second, by 78%; and in the third, by 94% (Fig. 1). This is very high activity taking into consideration the fact that oil does not contain phenol derivatives. It consisted of sesquiterpene hydrocarbons, which differed in stability. The dynamics of changes in the main components of the essential oil of ginger for the system with an oil concenVol. 45 No. 6 2009

80 5 70 4

60

30

60 days

Fig. 2. Content of polysildes (% from initial) in essential oils of garlic in the oxidation process in hexane solution: (1) diallyldisulde, (2) methylallyldisulde, (3) diallyltrisulde, (4) methylallyltrisulde, and (5) diallyltetrasulde.

mal content of cinnamon leaves, ginger, laurel, mace, and nutmeg oils (3.3 l/ml) had AOA more than 50%. The increase in the content of these oils by ve times was accompanied by an increase in AOA by 2030%, and the further increase in content of ginger and mace oils by four times (from 16.5 to 70 l/ml) led to the growth of oil activity by only 57%. The diluted solutions (3.3 l/ml) of juniper berry, black and white pepper, fennel, caraway, and cardamom oils had very low AOA: 2028%. The increase in concentration of such oils by ve times (from 3.3 to 16.5 l/ml) led to an increase in AOA by 2.53 times. The further increase in concentration of essential oils up to 70 l/ml either did not change AOA, as for juiper berry and black pepper oils, or it led to an increase by 710%. The stable increase in AOA from 21 to 45% and 66% with an increase in oil concentration by ve and four times was revealed only for cardamom oil (Fig. 1). The essential oil of garlic possesses the highest AOA (90%) and this activity depended poorly on oil concentration (Fig. 1). This oil was a mixture of polysuldes with methyl and allyl substituents. To assess changes in the content of the main oil components in the process of oxidation or storage, we calculated the ratio of quantity of each component of the oxidizing system to its initial quantity, which was regarded as 100%. This allowed us to assess the extent of oxidation of the component or the increase in its content in case this component was the nal oxidation product of other essential oil components. Figure 2 presents

APPLIED BIOCHEMISTRY AND MICROBIOLOGY

ANTIOXIDANT PROPERTIES OF ESSENTIAL OILS % 120 % 100 I 80 5 80 4 3 60 2 40 0 20 40 60 days 20 40 60 II

645

100

Fig. 3. Content of main components (% from initial) of essential oil of ginger in the oxidation process in hexane solution: (1) -curcumene, (2) zingiberene, (3) -bisabolene, (4) -bisabolene, and (5) sesquiphellandrene.

tration 16.5 l/ml is presented in Fig. 3. The main oil componentzingibereneoxidizes faster than all others. After 60 days of storage, its content in the rst solution decreased by 20 times, in the second it decreased by seven times, and in the third it decreased by 2.5 times. Also, the content of bisabolenes and sesquiphellandrene decreased (Fig. 3). It is interesting that, in parallel to the decrease in zingiberene concentration, we saw an increase in -curcumene content. The structure of zingiberene is similar to the structure of -terpinene. Both compounds have a similar hexamerous cycle with two double bonds. The oxidation product of -terpinene was p-cymen [2729]. Probably, zingiberene oxidized in -curcumen, which is an aromatic compound similar to p-cymen. The content of zingiberene in ginger oil is approximately 35%, and thus this oil has high AOA. During storage of the essential oil of ginger away from light for 12 months, the content of zingiberene decreased by two times and the content of -curcumen increased by 1.5 times. Such a change in the oil composition signicantly worsens its organoleptic characteristics and physicochemical and biological properties. This should be taken into consideration when using stored essential oil of ginger. The essential oil of sweet marjoram contained from 15 to 25% linalool, 4-terpineol, and -terpinene and did not contain phenol derivatives. With the increase in oil concentration, its AOA increased by 1.5 times and constituted 72% (Fig. 1). Figure 4 presents the quantity of compounds left after 40 days of oxidation (in percentage from the initial quantity). During the storage of the diluted solution of oil, mono- and sesquiterpene hydrocarbons and even 4-terpeneol were signicantly oxidized; in a more concentrated solution, only the contentof - and -terpenenes decreased signicantly. The content of oxidation products of these comAPPLIED BIOCHEMISTRY AND MICROBIOLOGY

Fig. 4. Inuence of concentration of marjoram oil (I 3.3 l/ml, II16.5 l/ml) on the degree of oxidation of its components (% of initial): (1) sabinene, (2) myrcene, (3) terpinene, (4) -terpinene, (5) -terpinolene, (6) -caryophyllene, (7) geraniol, and (8) 4-terpineol.

pounds, p-cymene, increased. In both solutions the content of geraniol changed slightly. The individual marjoram oil preserved content stability away from light for a year; in light, noticeable oxidation began after 4 months of storage [28]. Black and white pepper and juniper berry oils demonstrated a signicant inuence of the concentration on antioxidant properties. They had practically the same quantitative composition of componentsmono- and disesquiterpene hydrocarbons. In hexane solution they did not show antioxidant properties because the oxidation of 2-hexenal happened to the same extent as in the control sample, which did not contain essential oils. In a concentration 16.5 l/ml, the essential oil of black pepper hindered the oxidation of 2-hexenal by 58%, the essential oil of white pepper hindered it by 49%, and juniper berry oil hindered it by 65%. In a solution with a concentration of 70 l/ml, the essential oil of black pepper had an AOA of 62% and that of juniper berry, 66% (Fig. 1). In diluted solutions, during 40 days, nearly all components of the essential oil were oxidized. Figure 5 shows the changes in the content of the main oil components for a solution with a concentration of 16.5 l/ml. As is seen, after 40 days the content of and -terpinenes in oils decreased by ve to six times and that of -cariophyllene, by 2.53 times; the content of other components changed insignicantly. We have shown that the oxidation of caryophyllene began when the majority of - and -terpinenes were oxidized. During storage of individual oils away from light, a similar process was detected after 8 months. By that time, the
No. 6 2009

Vol. 45

646 % 100 80 60 40 20 0 I II III

MISHARINA et al. % 100 4 90 3 80 70 2 60 50 40 1 1 2 3 4 5 6 7 30 0 20 40 60 100 days

Fig. 5. Content of main components (% of initial) in (I) black pepper, (II) white pepper, and (III) juniper berry in the oxidation process in hexane solution: (1) - and -pinenes, (2) sabinene, (3) myrcene, (4) 3-carene, (5) is limonene, (6) - and -terpinenes, and (7) -caryophyllene.

Fig. 6. Content of sabinene and terpinyl acetate (% of initial) in cardamom oil and its mixture with 0.5% clove bud oil during storage: (1) and (2) sabinene and terpinyl acetate in cardamom oil, and (3) and (4) sabinene and terpinyl acetate in a mixture of cardamom oil and 0.5% clove bud oil.

content of terpinenes was 210% of the initial content and that of caryophyllene was 8085%, whereas the content of caryophyllene oxide increased by 2 2.5 times and that of p-cymene increased by four times. Thus, the content of -terpinene and caryophyllene oxide may be an indicator of the freshness and quality of the essential oils of black and white pepper. Additional experiments showed that the stability of these oils is much higher than in the mixture of, for example, black pepper, juniper berry, garlic, or nutmeg. With the increase in fennel and caraway essential oils concentration in hexane solution, their AOA also increased by 2.73 times and was 60% for fennel and 56% for caraway. In a solution with an oil concentration of 70 l/ml, AOA was 71 and 67%, respectively (Fig. 1). In a diluted solution of fennel oil, trans-anetol oxidized completely after 40 days to an anise aldehyde; with an increase in concentration, the degree of oxidation decreased and constituted 50% at a concentration of 16.5 l/ml and 46% at a concentration 70 l/ml. A similar oxidation of anetol to anise aldehyde was discovered in a study of the stability of fennel oil in light and away from light [29]. In caraway oil, limonene was unstable. In a diluted solution after 40 days, only 6% of it was left; of carvon only 51% was left; in more concentrated solutions, limonene remained at 7882% and carvon remained at 9495%. We showed that in the mixture of fennel, laurel, and coriander oils, the oxidation of the oil components, including limonene and anetol, did not happen and this mixture remained stable for 12 months [29]. The essential oils of laurel and cardamom had close compositions of the main components; the difference in

aromas was due to the presence in cardamom oil of 1% of neryl and linalyl acetates and nerolidol. Both oils contain sabinene and 1,8-cineol, but the essential oil of laurel in hexane solution hindered the oxidation of 2-hexenal 1.5 times more effectively than the essential oil of cardamom (Fig. 1). After 40 days of autooxidation in light in hexane solution, laurel oil remained stable. Earlier we had established that individual pure laurel oil did not change its content in storage away from light for 2 years [29]. The stability of the essential oil of cardamom, as well as its AOA, depended on its concentration in hexane solution. So, at a concentration of 3.3 l/ml, The quantity of sabinene decreased by ve times and terpinyl acetate decreased by two times, and -terpinenes oxidized completely. In more concentrated solutions, only - and -terpinenes oxidized completely; the content of other components in solutions with oil concentrations 16.5 and 70 l/ml changed in the same way. It is noteworthy that the AOA of cardamom oil increased with increasing concentration in the model system (Fig. 1). Probably, they were mainly - and -terpinenes, which corresponded to the AOA properties of this oil. During storage of the essential oil of cardamom, after 100 days we noticed signicant oxidation of - and -terpinenes; the quantity of sabinene decreased by 65% and that of terpinyl acetate decreased by 35% (Fig. 6). The reason for changes in the behavior of essential oils of laurel and cardamom was probably that laurel oil contained approximately 2% of methyl eugenol, which possesses strong antioxidation properties. Due to its presence, the laurel oil has higher AOA and was resistant to oxidation. Addition to the cardamom essential oil at least 0.5% of clove bud oil, which
Vol. 45 No. 6 2009

APPLIED BIOCHEMISTRY AND MICROBIOLOGY

ANTIOXIDANT PROPERTIES OF ESSENTIAL OILS

647

contained approximately 80% eugenol, signicantly increased the resistance of all components to oxidation in comparison with individual cardamom oil (Fig. 6). Thus, our research and the literature data show that essential oils are effective natural antioxidants, which are able to compete with synthetic ones. The antioxidant properties of essential oils are determined by their composition. Oils with high contents of substituted phenols are able to signicantly hinder the oxidation processes of labile unsaturated aldehydes even in low concentrations. The antioxidant properties of essential oils consisting of terpene hydrocarbons and alcohols are determined by - and -terpinenes and their sesquiterpene analogs. We revealed a signicant inuence of the concentration of such oils on their antioxidant properties. It was established that in the oxidation of the main components of essential oils, substances are formed that are present in natural essential oils. The stability of the composition of essential oils increased with an increase in their concentrations in model solutions. The oxidation of substances in pure essential oils happened slower than in solutions. REFERENCES
1. Madsen, L.H., Nielsen, B.R., Bertelsen, G., and Skibsted, L.H., Food Chem., 1996, vol. 57, pp. 331337. 2. Voitkevich, S.A., in Ernye masla dlya parfyumerii i aromaterapii (Essential Oils for Perfumery and Aromatherapy), Moscow: Pishch. Prom., 1999. 3. Flavours and Fragrances. Chemistry, Bioprocessing and Sustainability, Berger, R.G., Ed., New York: Springer, 2007, pp. 43116. 4. Bauer, K., Garbe, D., and Surburg, H., Common Fragrance and Flavor Materials, Weinheim: VCH Verlag, 1990. 5. Cervato, G., Carabelli, M., Gervasio, S., Cittera, A., Cazzola, R., and Cestaro, B., J. Food Biochem., 2000, vol. 24, no. 3, pp. 453465. 6. Dorman, H.J.D., Figueiredo, A.C., Barroso, J.G., and Deans, S.G., Flavour Fragrances J, 2000, vol. 15, no. 1, pp. 1215. 7. Lee, K.G. and Shibamoto, T., Food Chem., 2001, vol. 74, pp. 443448. 8. Lee, K.W., Kim, Y.J., Kim, D.-O., Lee, H.J., and Lee, C.Y., J. Agric. Food Chem., 2003, vol. 51, no. 22, pp. 65166520. 9. Bowry, V.W. and Ingold, K.U., Acc. Chem. Res., 1999, vol. 32, no. 1, pp. 2734.

10. Ruberto, G. and Baratta, M., Food Chem., 2002, vol. 69, pp. 167174. 11. Foti, M.C. and Ingold, K.U., J. Agric. Food Chem., 2003, vol. 51, no. 9, pp. 27582765. 12. Ruberto, G., Baratta, M.T., Deans, S.G., and Dorman, H.J.D., Planta Med., 2000, vol. 66, no. 3, pp. 687690. 13. El-Ghorab, A.H., Mansour, A.F., and El-Massry, K.F., Flavour Fragrance J., 2004, vol. 19, no. 3, pp. 5458. 14. Kim, H.J., Chen, F., Wu, C.Q., Wang, X., Chung, H.Y., and Jin, Z.Y., J. Agric. Food Chem., 2004, vol. 52, no. 7, pp. 24852488. 15. Singh, G., Marimuthu, R., De Heluani, C.S., and Catalan, C., J. Food Sci., 2005, vol. 70, no. 3, pp. 141144. 16. Wei, A. and Shibamoto, T., J. Agric. Food Chem., 2007, vol. 55, no. 5, pp. 17371742. 17. Menut, C., Bessiere, J.M., Samate, D., Djibo, A.K., Buchbauer, G., and Schopper, B., J. Essent. Oil Res., 2000, vol. 12, no. 1, pp. 207211. 18. Minica-Dukic, N., Bozin, B., Sokovic, M., and Simin, N., J. Agric. Food Chem., 2004, vol. 52, no. 7, pp. 24852489. 19. Agnanient, H., Makani, T., Akagah, A., Menut, C., and Bessiere, J.M., Flavour Fragrance J., 2002, vol. 20, no. 1, pp. 3437. 20. Lee, K.G. and Shibamoto, T., J. Agric. Food Chem., 2002, vol. 50, no. 15, pp. 49474952. 21. Lee, K.G. and Shibamoto, T., Food Chem. Toxicol., 2001, vol. 39, no. 10, pp. 11991204. 22. Yanagimoto, K., Ochi, H., Lee, K.G., and Shibamoto, T., J. Agric. Food Chem., 2003, vol. 51, no. 25, pp. 7396 7401. 23. Lee, S.J., Shibamoto, T., and Lee, K.G., Food Chem., 2005, vol. 91, pp. 131137. 24. Bozin, B., Milica-Dukic, N., Simin, N., and Anachov, G., J. Agric. Food Chem., 2006, vol. 54, no. 5, pp. 18221828. 25. Misharina, T.A. and Samusenko, A.L., Prikl. Biokhim. Mikrobiol., 2008, vol. 44, no. 4, pp. 482486. 26. Jennings, W. and Shibamoto, T., Qualitative Analysis of the Flavor and Fragrance Volatiles by Glass Capillary Gas Chromatography, New York: Academic, 1980, pp. 130154. 27. Misharina, T.A., Prikl. Biokhim. Mikrobiol., 2001, vol. 37, no. 6, pp. 726732. 28. Misharina, T.A., Polshkov, A.N., Ruchkina, E.L., and Medvedeva, I.B., Prikl. Biokhim. Mikrobiol., 2003, vol. 39, no. 3, pp. 353358. 29. Misharina, T.A. and Polshkov, A.N., Prikl. Biokhim. Mikrobiol., 2005, vol. 41, no. 6, pp. 693702.

APPLIED BIOCHEMISTRY AND MICROBIOLOGY

Vol. 45

No. 6

2009